C-terminal PDZ Binding Domain Research Articles

Expression profile, subcellular localization of MARCH4 and transcriptome analysis of its potential regulatory signaling pathway in large yellow croaker (Larimichthys crocea)

Membrane-associated RING-CH (MARCH) family, as Ring-type E3 ligases, have attracted extensive attention to their immune functions. MARCH4 plays an essential role in regulating immune response in mammal. In the present study, it is the first to report on MARCH4 characteristics and signal pathway in fish. MARCH4 in large yellow croaker Larimichthys crocea (named as LcMARCH4) encodes a RING-CH domain and two TM domains, as well as other function domains, including an N-terminal proline rich domain, an AxxxG-motif in TM1, a tyrosine-based YXXØ motif, and a C-terminal PDZ-binding domain. LcMARCH4 is a tissue-specific protein with highly significant expression in brain. The mRNA transcripts of LcMARCH4 were significantly induced in the main organs (skin, gill, spleen, and head-kidney) by C. irritans infection. Consistently, significant increase was observed in spleen and head-kidney after LPS, Poly I:C stimulation and V. parahaemolyticus infection. Subcellular localization analysis showed that LcMARCH4 was localized in the cytoplasm and membrane. Moreover, we found 46 DEGs in a comparative transcriptome analysis between the LcMARCH4 overexpression group and control vector group. The analysis showed that HSPA6, HSPA1B and DNAJB1 might play important regulatory roles to MARCH4 in fish. Notably, two noncoding RNA, both RN7SL1 and RN7SL2, the expression levels went up in MARCH4 overexpression cells. Taken together, this study will provide new insights into finfish MARCH4 and its potential regulatory signaling pathway as well.

Multimerization and Retention of the Scavenger Receptor SR-B1 in the Plasma Membrane

Scavenger receptor B1 (SR-B1), the main receptor for high-density lipoprotein (HDL), is key in preventing atherosclerosis. It removes cholesterol from HDL, returning the lipid-poor lipoprotein to the circulation. To study the mechanisms controlling SR-B1 dynamics at the plasma membrane and its internalization rate, we developed a single-chain variable fragment (ScFv) antibody to image the receptor in live cells and track the behavior of single SR-B1 molecules. Unlike transferrin receptors, cholera-toxin-binding gangliosides, and bulk membrane markers, SR-B1 was internalized only marginally over hours. Plasmalemmal retention was not attributable to its C-terminal PDZ-binding domain or to attachment to the cortical cytoskeleton. Instead, SR-B1 undergoes multimerization into large metastable clusters that, despite being mobile in the membrane, fail to enter endocytic pathways. SR-B1 multimerization was impaired by mutating its C-terminal leucine zipper and by disrupting actin polymerization, causing rapid receptor internalization. Multimerization and plasmalemmal retention are critical for SR-B1 function.

Nav1.5 N-terminal domain binding to α1-syntrophin increases membrane density of human Kir2.1, Kir2.2 and Nav1.5 channels.

Cardiac excitability and refractoriness are largely determined by the function and number of inward rectifier K⁺ channels (Kir2.1-2.3), which are differentially expressed in the atria and ventricles, and Nav1.5 channels. We have focused on how Nav1.5 and Kir2.x function within a macromolecular complex by elucidating the molecular determinants that govern Nav1.5/Kir2.x reciprocal modulation. The results demonstrate that there is an unexpected 'internal' PDZ-like binding domain located at the N-terminus of the Nav1.5 channel that mediates its binding to α1-syntrophin. Nav1.5 N-terminal domain, by itself (the 132 aa peptide) (Nter), exerts a 'chaperone-like' effect that increases sodium (I(Na)) and inward rectifier potassium (I(K1)) currents by enhancing the expression of Nav1.5, Kir2.1, and Kir2.2 channels as demonstrated in Chinese hamster ovary (CHO) cells and in rat cardiomyocytes. Site-directed mutagenesis analysis demonstrates that the Nter chaperone-like effect is determined by Serine 20. Nav1.5-Kir2.x reciprocal positive interactions depend on a specific C-terminal PDZ-binding domain sequence (SEI), which is present in Kir2.1 and Kir2.2 channels but not in Kir2.3. Therefore, in human atrial myocytes, the presence of Kir2.3 isoforms precludes reciprocal I(K1)-INa density modulation. Moreover, results in rat and human atrial myocytes demonstrate that binding to α1-syntrophin is necessary for the Nav1.5-Kir2.x-positive reciprocal modulation. The results demonstrate the critical role of the N-terminal domain of Nav1.5 channels in Nav1.5-Kir2.x-reciprocal interactions and suggest that the molecular mechanisms controlling atrial and ventricular cellular excitability may be different.

Abstract 4408: Her2 C terminal PDZ binding domain interacting with DLG1 is required for radioresistance through induction of EMT and loss of epithelial polarity

Abstract The tyrosine kinase receptor HER2 (ErbB2/neu) is predominantly localized to the basolateral membrane of the epithelium and has been implicated in mammary tumorigenesis as well as in resistance to chemotherapy and radiation. Here we demonstrate that HER2 interacts with discs large 1 (DLG1) that is one of PDZ domain proteins expressed at the basolateral membrane of human breast epithelial cells and plays an important role in the establishment and maintenance of epithelial polarity in Drosophila. However, either loss of the last 6 C-terminal amino acids (HER2-Δ1250-1255) or a valine-to-alanine C-terminal substitution (HER2-V1255A) in HER2, abrogates the interaction with DLG1. In contrast, either PDZ domain 1 or 3 within Dlg1 is required for interaction with HER2. Moreover, HER2-Δ1250-1255- or HER2-V1255A-expressing MCF10A human breast epithelial cells show the reduction of mesenchymal-to-epithelial transition (EMT) proteins including vimentin and Snail. In addition, HER2-wt cells promotes EMT as shown by down-regulation of E-cadherin and upregulation of vimentin and Snail. Taken together, these results indicate that C-terminal PDZ binding domain in HER2 modulates biological functions of HER2. These results reveal new insight into the structure and function of HER2, which may guide the design and development of new anti-cancer targets to treat HER2-positive breast cancer. Citation Format: Dong Lin, Xiaodi Zhang, Angela Eldridge, Ming Fan, Cheikh Menaa, Jianjian Li. Her2 C terminal PDZ binding domain interacting with DLG1 is required for radioresistance through induction of EMT and loss of epithelial polarity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4408. doi:10.1158/1538-7445.AM2014-4408

Intracellular and extracellular domains of protein tyrosine phosphatase PTPRZ-B differentially regulate glioma cell growth and motility.

Gliomas are primary brain tumors for which surgical resection and radiotherapy is difficult because of the diffuse infiltrative growth of the tumor into the brain parenchyma. For development of alternative, drug-based, therapies more insight in the molecular processes that steer this typical growth and morphodynamic behavior of glioma cells is needed. Protein tyrosine phosphatase PTPRZ-B is a transmembrane signaling molecule that is found to be strongly up-regulated in glioma specimens. We assessed the contribution of PTPRZ-B protein domains to tumor cell growth and migration, via lentiviral knock-down and over-expression using clinically relevant glioma xenografts and their derived cell models. PTPRZ-B knock-down resulted in reduced migration and proliferation of glioma cells in vitro and also inhibited tumor growth in vivo. Interestingly, expression of only the PTPRZ-B extracellular segment was sufficient to rescue the in vitro migratory phenotype that resulted from PTPRZ-B knock-down. In contrast, PTPRZ-B knock-down effects on proliferation could be reverted only after re-expression of PTPRZ-B variants that contained its C-terminal PDZ binding domain. Thus, distinct domains of PTPRZ-B are differentially required for migration and proliferation of glioma cells, respectively. PTPRZ-B signaling pathways therefore represent attractive therapeutic entry points to combat these tumors.

SUMO, PTEN and Tumor Suppression.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was identified in 1997 as a tumor suppressor gene through mapping of homozygous mutations occurring in multiple sporadic tumor types and in patients with cancer predisposition syndromes including Cowden disease (Song et al., 2012). Since that time, PTEN has emerged as one of the most frequently mutated or deleted genes in human cancers, including human skin cancers. In particular, loss of PTEN function through mutation or deletion has been observed in up to 70% of melanoma cell lines, and epigenetic silencing of PTEN has been observed in 30-40% of malignant melanomas (Mehnert and Kluger, 2012).

Angiomotin-like2 Gene (amotl2) Is Required for Migration and Proliferation of Endothelial Cells during Angiogenesis

Angiogenesis involves sprouting, migration, and proliferation of endothelial cells. The angiomotin-like2 gene (amotl2) has been found in blood vessels in zebrafish embryos, but its function in angiogenesis and underlying mechanisms remain unknown. In this study, we demonstrate that knockdown of amotl2 in zebrafish Tg(fli1:EGFP)(y1) and Tg(fli1:nEGFP)(y7) transgenic embryos impairs the intersegmental vessel growth and suppresses proliferation of endothelial cells. Transplantation experiments indicate that function of amotl2 in intersegmental vessel growth is cell-autonomous. AMOTL2 knockdown in cultured human umbilical vein endothelial cells also inhibits cell proliferation and migration and disrupts cell polarity, ultimately interrupting the formation of vascular tube-like structures. Amotl2 promotes MAPK/ERK activation via c-Src, which is dependent on phosphorylation of tyrosine residue at position 103 but independent of the C-terminal PDZ-binding domain. Taking together, our data indicate that Amotl2 plays a pivotal role in polarity, migration and proliferation of angiogenic endothelial cells.

A C-terminal PDZ binding domain modulates the function and localization of Kv1.3 channels

The voltage-gated potassium channel, Kv1.3, plays an important role in regulating membrane excitability in diverse cell types ranging from T-lymphocytes to neurons. In the present study, we test the hypothesis that the C-terminal PDZ binding domain modulates the function and localization of Kv1.3. We created a mutant form of Kv1.3 that lacked the last three amino acids of the C-terminal PDZ-binding domain (Kv1.3ΔTDV). This form of Kv1.3 did not bind the PDZ domain containing protein, PSD95. We transfected wild type and mutant Kv1.3 into HEK293 cells and determined if the mutation affected current, Golgi localization, and surface expression of the channel. We found that cells transfected with Kv1.3ΔTDV had greater current and lower Golgi localization than those transfected with Kv1.3. Truncation of the C-terminal PDZ domain did not affect surface expression of Kv1.3. These findings suggest that PDZ-dependent interactions affect both Kv1.3 localization and function. The finding that current and Golgi localization changed without a corresponding change in surface expression suggests that PDZ interactions affect localization and function via independent mechanisms.

Arrestin and the Multi-PDZ Domain-containing Protein MPZ-1 Interact with Phosphatase and Tensin Homolog (PTEN) and Regulate Caenorhabditis elegans Longevity

Arrestins are multifunctional adaptor proteins best known for their role in regulating G protein-coupled receptor signaling. Arrestins also regulate other types of receptors, including the insulin-like growth factor receptor (IGF-1R), although the mechanism by which this occurs is not well understood. In Caenorhabditis elegans, the IGF-1R ortholog DAF-2 regulates dauer formation, stress resistance, metabolism, and lifespan through a conserved signaling cascade. To further elucidate the role of arrestin in IGF-1R signaling, we employed an in vivo approach to investigate the role of ARR-1, the sole arrestin ortholog in C. elegans, on longevity. Here, we report that ARR-1 functions to positively regulate DAF-2 signaling in C. elegans. arr-1 mutant animals exhibit increased longevity and enhanced nuclear localization of DAF-16, an indication of decreased DAF-2 signaling, whereas animals overexpressing ARR-1 have decreased longevity. Genetic and biochemical analysis reveal that ARR-1 functions to regulate DAF-2 signaling via direct interaction with MPZ-1, a multi-PDZ domain-containing protein, via a C-terminal PDZ binding domain in ARR-1. Interestingly, ARR-1 and MPZ-1 are found in a complex with the phosphatase and tensin homolog (PTEN) ortholog DAF-18, which normally serves as a suppressor of DAF-2 signaling, suggesting that these three proteins work together to regulate DAF-2 signaling. Our results suggest that the ARR-1-MPZ-1-DAF-18 complex functions to regulate DAF-2 signaling in vivo and provide insight into a novel mechanism by which arrestin is able to regulate IGF-1R signaling and longevity.

Normal Hepatic Cell Surface Localization of the High Density Lipoprotein Receptor, Scavenger Receptor Class B, Type I, Depends on All Four PDZ Domains of PDZK1

PDZK1 is a four PDZ domain-containing scaffold protein that binds to scavenger receptor class B, type I (SR-BI), the high density lipoprotein receptor, by its first PDZ domain (PDZ1). PDZK1 knock-out mice exhibit a >95% decrease in hepatic SR-BI protein and consequently an approximately 70% increase in plasma cholesterol in abnormally large high density lipoprotein particles. These defects are corrected by hepatic overexpression of full-length PDZK1 but not the PDZ1 domain alone, which partially restores SR-BI protein abundance but not cell surface expression or function. We have generated PDZK1 knock-out mice with hepatic expression of four PDZK1 transgenes encoding proteins with nested C-terminal truncations: pTEM, which lacks the three C-terminal residues (putative PDZ-binding motif), and PDZ1.2, PDZ1.2.3, or PDZ1.2.3.4, which contain only the first two, three, or four N-terminal PDZ domains, respectively, but not the remaining C-terminal sequences. Hepatic overexpression of pTEM restored normal hepatic SR-BI abundance, localization, and function. Hepatic overexpression of PDZ1.2 or PDZ1.2.3 partially restored SR-BI abundance ( approximately 12 or approximately 30% of wild type, respectively) but did not (PDZ1.2) or only slightly (PDZ1.2.3) restored hepatic SR-BI cell surface localization and function. Hepatic overexpression of PDZ1.2.3.4 completely restored SR-BI protein abundance, cell surface expression, and function (normalization of plasma cholesterol levels). Thus, all four PDZ domains in PDZK1, but not PDZ1-3 alone, are sufficient for its normal control of the abundance, localization, and therefore function of hepatic SR-BI, whereas the residues C-terminal to the PDZ4 domain, including the C-terminal putative PDZ-binding domain, are not required.

A lysophosphatidic acid receptor lacking the PDZ-binding domain is constitutively active and stimulates cell proliferation

Lysophosphatidic acid (LPA) is an extracellular signaling lipid that regulates cell proliferation, survival, and motility of normal and cancer cells. These effects are produced through G protein–coupled LPA receptors, LPA 1 to LPA 5. We generated an LPA 1 mutant lacking the SerValVal sequence of the C-terminal PDZ-binding domain to examine the role of this domain in intracellular signaling and other cellular functions. B103 neuroblastoma cells expressing the mutant LPA 1 showed rapid cell proliferation and tended to form colonies under serum-free conditions. The enhanced cell proliferation of the mutant cells was inhibited by exogenous expression of the plasmids inhibiting G proteins including G βγ, G αi and G αq or G α12/13, or treatment with pertussis toxin, phosphoinositide 3-kinase (PI3K) inhibitors or a Rho inhibitor. We confirmed that the PI3K-Akt and Rho pathways were intrinsically activated in mutant cells by detecting increases in phosphorylated Akt in western blot analyses or by directly measuring Rho activity. Interestingly, expression of the mutant LPA 1 in non-tumor mouse fibroblasts induced colony formation in a clonogenic soft agar assay, indicating that oncogenic pathways were activated. Taken together, these observations suggest that the mutant LPA 1 constitutively activates the G protein signaling leading to PI3K-Akt and Rho pathways, resulting in enhanced cell proliferation.

The CD34-Related Molecule Podocalyxin Is a Potent Inducer of Microvillus Formation

BackgroundPodocalyxin is a CD34-related transmembrane protein involved in hematopoietic cell homing, kidney morphogenesis, breast cancer progression, and epithelial cell polarization. Although this sialomucin has been shown to block cell adhesion, the mechanisms involved remain enigmatic. It has, however, been postulated that the adaptor proteins NHERF-1 and 2 could regulate apical targeting of Podocalyxin by linking it to the actin cytoskeleton.Principal FindingsHere, in contrast, we find that full-length Podocalyxin acts to recruit NHERF-1 to the apical domain. Moreover, we show that ectopic expression of Podocalyxin in epithelial cells leads to microvillus formation along an expanded apical domain that extends laterally to the junctional complexes. Removal of the C-terminal PDZ-binding domain of Podocalyxin abolishes NHERF-1 recruitment but, surprisingly, has no effect on the formation of microvilli. Instead, we find that the extracellular domain and transmembrane region of Podocalyxin are sufficient to direct recruitment of filamentous actin and ezrin to the plasma membrane and induce microvillus formation.Conclusions/SignificanceOur data suggest that this single molecule can modulate NHERF localization and, independently, act as a key orchestrator of apical cell morphology, thereby lending mechanistic insights into its multiple roles as a polarity regulator, tumor progression marker, and anti-adhesin.

Asymmetric Localization of Vangl2 and Fz3 Indicate Novel Mechanisms for Planar Cell Polarity in Mammals

Planar cell polarity (PCP) is a process in which cells develop with uniform orientation within the plane of an epithelium. To begin to elucidate the mechanisms of PCP in vertebrates, the localization of the protein Vangl2 (Van Gogh-like) was determined during the development of the mammalian cochlea. Results indicate that Vangl2 becomes asymmetrically localized to specific cell-cell boundaries along the axis of polarization and that this asymmetry is lost in PCP mutants. In addition, PDZ2 (postsynaptic density/Discs large/zona occludens 1), PDZ3, and PDZ4 of the PCP protein Scrb1 (Scribble) are shown to bind to the C-terminal PDZ binding domain of Vangl2, suggesting that Scrb1 plays a direct role in asymmetric targeting of Vangl2. Finally, Fz3 (Frizzled), a newly demonstrated mediator of PCP, is also asymmetrically localized in a pattern that matches that of Vangl2. The presence and asymmetry of Fz3 at the membrane is shown to be dependent on Vangl2. This result suggests a role for Vangl2 in the targeting or anchoring of Fz3, a hypothesis strengthened by the existence of a physical interaction between the two proteins. Together, our data support the idea that protein asymmetry plays an important role in the development of PCP, but the colocalization and interaction of Fz3 and Vangl2 suggests that novel PCP mechanisms exist in vertebrates.

Separation Force Measurements Reveal Different Types of Modulation of E-cadherin-based Adhesion by Nectin-1 and -3

Nectins are Ca2+-independent cell adhesion molecules found at cadherin-based adherens junctions. We used a dual pipette assay that measures the forces required to separate cell doublets to determine how nectins affect the formation and strength of cell-cell adhesion. Less force was required to separate doublets of L cells expressing nectin-1 or nectin-3 than to separate doublets of E-cadherin-expressing cells. Heterodimers formed between cells expressing nectin-1 or nectin-3 adhered more strongly than homodimers. Nectin-3 that does not trans-interact with nectin-1 inhibited E-cadherin-mediated adhesion. However, the extracellular fragment of nectin-1 did not have an agonistic effect on E-cadherin-dependent cell adhesion when it trans-interacted with nectin-3, expressed at high levels in cells. In contrast, the extracellular fragment of nectin-3 had a significant agonistic effect on cadherin-based adhesion when it interacted with endogenous nectin-1, expressed at low levels in cells. Our results indicate that E-cadherin is the key molecule involved in cell adhesion and that the regulation of E-cadherin-based adhesion involving cellular nectin-1 trans-interacting with nectin-3 is qualitatively different from that involving cellular nectin-3 trans-interacting with nectin-1 and depends on the nectin levels expressed by cells.

Apactin is involved in remodeling of the actin cytoskeleton during regulated exocytosis

Apactin is an 80-kDa type I membrane glycoprotein derived from pro-Muclin, a precursor that also gives rise to the zymogen granule protein Muclin. Previous work showed that apactin is efficiently removed from the regulated secretory pathway and targeted to the actin-rich apical plasma membrane of the pancreatic acinar cell. The cytosolic tail (C-Tail) of apactin consists of 16 amino acids, has Thr casein kinase II and Ser protein kinase C phosphorylation sites, and a C-terminal PDZ-binding domain. Secretory stimulation of acinar cells causes a decrease in Thr phosphorylation and an increase in Ser phosphorylation of apactin. Fusion peptides of the C-Tail domain pulldown actin, ezrin, and EBP50/NHERF in a phosphorylation-dependent manner. HIV TAT-C-Tail fusion peptides were used as dominant negative constructs on living pancreatic cells to study effects on the actin cytoskeleton. During secretory stimulation, TAT-C-Tail-Thr/Asp phosphomimetic peptide caused an increase in actin-coated zymogen granules at the apical surface, while TAT-C-Tail-S/D phosphomimetic peptide caused a broadening of the actin cytoskeleton. These data indicate that stimulation-mediated Thr dephosphorylation allows decreased association of apactin with EBP50/NHERF and fosters actin remodeling to coat zymogen granules. Stimulation-mediated Ser phosphorylation increases apactin association with the actin cytoskeleton, maintaining tight bundling of actin microfilaments at the apical surface. Thus, apactin is involved in remodeling the apical cytoskeleton during regulated exocytosis in a manner controlled by phosphorylation of the apactin C-Tail.

Regulation of sodium transport in mammalian collecting duct cells by aldosterone-induced kinase, SGK1: structure/function studies

Serum- and glucocorticoid-induced kinases (SGK) are members of the serine–threonine kinase family. SGK1, the isoform identified first, is rapidly induced by aldosterone. In this study, we determined that the two recently described isoforms, SGK2 and SGK3 are also expressed in renal cortical collecting duct (CCD) cells; however, their expression is not induced by aldosterone or glucocorticoids. SGK1 increases the activity of the epithelial sodium channel (ENaC) in oocytes but its cellular targets in native mineralocorticoid target cells and its mechanism of action are still unknown. We studied the role of SGK1 in corticosteroid-regulated Na transport in M-1 mouse CCD cell lines that stably over-express or down-regulate SGK1. Basal rates of transepithelial Na transport were significantly lower in CCD cells in which SGK1 expression or activity was down-regulated than in SGK1 overexpressing cells. Importantly, corticosteroid treatment failed to stimulate Na transport in cells with down-regulated SGK1 while it significantly increased Na transport in parent and SGK1 overexpressing M-1 cells. To determine if C-terminal PDZ interactions are important for SGK’s effect on ENaC activity or trafficking, we examined the effects of mutant SGK1 in which the conserved PDZ binding domain has been eliminated. However, such mutations did not decrease its stimulatory effect on ENaC current in Xenopus oocytes. Fluorescence confocal microscopy revealed that the intracellular localization of full-length and PDZ binding mutated SGK1 was identical: they both localize to intracellular vesicular structures. On the other hand, N-terminally truncated (delta 60)-SGK1 did not increase ENaC activity. We conclude that SGK1 is a critical component in corticosteroid-regulated Na transport in mammalian CCD cells. Our data also indicate that the N-terminal of SGK1 is necessary for its stimulatory effect on Na transport while elimination of the C-terminal PDZ binding domain did not change its function.

EEG features associated with the occurrence of epilepsy after surgery for intracranial aneurysm and acoustic neuroma.

An investigation was carried out on 199 postoperative EEGs from 83 patients who underwent surgery for ruptured intracranial aneurysm or acoustic neuroma removal. The tracings were quantified without knowlege of the diagnostic group and whether or not epilepsy had supervened. The number of spikes and sharp components and slow waves at the site of the operative brain lesion were substantially and often significantly greater in the tracings from patients who developed epilepsy than from those who did not.